Immunological Reagents

ABSTRACT

This disclosure relates to binding agents with specificity for programmed cell death 1 (PD-1) and to methods for using the same to treat, prevent and/or ameliorate an infectious disease (e.g., human immunodeficiency virus (HIV)), cancer and/or autoimmunity.

RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/329,760 filed Jan. 27, 2017, now U.S. Pat. No. 9,982,053 B2, whichwas filed under 35 U.S.C. § 371 and claims priority to InternationalApplication No. PCT/IB2015/055943 filed Aug. 5, 2015, and claimspriority to U.S. Ser. No. 62/033,177 filed Aug. 5, 2014; U.S. Ser. No.62/053,366 filed Sep. 22, 2014; and U.S. Ser. No. 62/093,368 filed Dec.17, 2014; each of which is hereby incorporated by reference into thisdisclosure in their entirety.

FIELD OF THE DISCLOSURE

This disclosure relates to binding agents with specificity forprogrammed cell death 1 (PD-1) (e.g., human PD-1) and to methods forusing the same to treat and/or prevent infection (e.g., by humanimmunodeficiency virus (HIV)), cancer and/or autoimmunity.

BACKGROUND OF THE DISCLOSURE

As we enter the fourth decade of the HIV epidemic, significant advanceshave been made in the understanding of HIV pathogenesis and in thedevelopment of potent and safe antiviral drugs. More than 30 antiviraldrugs have been registered and the impact of combination antiretroviraltherapy (ART) on both morbidity and mortality has been remarkable.However, despite the long-term suppression of HIV replication achievedin patients with optimal adherence to ART, HIV invariably rebounds afterinterruption of therapy. Furthermore, successful therapy does not induceor allow restoration/development of virus-specific immune responsescapable of controlling HIV replication in the absence of ART. Thus,life-long ART is needed to control HIV replication and associateddisease in the large majority of HIV infected subjects.

A population of long-lived central memory CD4 T-cells latently infectedwith HIV has been identified in the blood as an important component ofthe HIV cell reservoir and as the primary cause of HIV persistence. Thelife-span of this latent cell reservoir is estimated to be approximately70 years in the presence of full HIV suppression with ART. However,recent studies have demonstrated that two populations of CD4 T-cellsresident in lymph nodes serve as the primary CD4 T-cell compartment forHIV infection, replication and production. These two CD4 T-cellpopulations are defined by the expression of PD-1 and CXCR5 and includethe PD-1+CXCR5+, i.e. T follicular helper cells (Tfh) and PD-1+CXCR5-CD4T-cell populations.

A number of mechanisms responsible for the establishment and maintenanceof the HIV latent cell reservoir(s) have been proposed. One of themechanisms is the persistent of minimal virus replication under ARTwhich may replenish the HIV cell reservoir. Therefore, ART is unable toinduce full suppression of HIV replication and the “natural” HIV-1specific immune response under ART is also unable to totally suppressand eliminate ongoing residual virus replication. The failures of ARTand of the HIV-specific immune response provide the rationale forinvestigating alternative interventions to target also the persistentHIV cell reservoir.

A number of immunological interventions have been investigated in thepast and currently being further developed with the goal to achieve HIVfunctional cure, wherein viral replication is suppressed withoutsustained antiviral therapy (9). Therapeutic vaccine strategies havebeen the primary intervention strategy investigated but the results haveshown modest efficacy in experimental animal models and patients withthe exception of a CMV-based vector HIV vaccine (50% efficacy in the NHPmodel;10). Recent studies have generated interesting results on thepossibility of using anti-envelope broad neutralizing antibodies (bNabs)as therapeutic agents in HIV infection (11,12). Furthermore, antagonistPD-1 Abs have been shown to restore T-cell functions in HIV infectedpatients and the possibility to use these Abs as a therapeutic strategyto augment the potency of HIV-specific T-cell responses has beenproposed (13,14).

It is well established that infiltrating tumor-specific CD8 T-cells aredysfunctional with regard their ability to proliferate and to mediatecytotoxic activity. The large majority of infiltrating tumor-specificCD8 T-cells are in a so-called exhaustion functional state. The primarymechanism responsible for the exhaustion of infiltrating tumor-specificCD8 T-cells is the increased expression of a number of regulatoryreceptors and particularly PD-1 regulatory receptor. The observationthat the blockade of the PD-1/PDL-½ (PD-1 ligands) is associated withthe recovery of CD8 T-cells from exhaustion has provided the rationalefor developing intervention strategies targeting the PD-1 moleculeexpressed by exhaustedCD8 T-cells. Recent studies have shown verypromising results with the use of PD-1 antibodies with antagonistactivity in patients with advanced cancer-associated disease. Studieshave show substantial rates of response, ranging from 18 to 40%, inpatients with advanced melanoma, non-small cell lung carcinoma and renalcarcinoma. Anti-PD-1 antibodies in these studies have been used eitheralone or in combination with an anti-CTL-A4 antibody. After theseinitial studies, the current studies are being performed in patientswith a variety of tumors including also hematological tumors.

There is a need in the art for additional reagents for targeting PD-1and methods for using the same. This disclosure addresses those needs byproviding reagents and methods that may be used to target PD-1 and cellsand/or tissues expressing the same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1A. Mouse PD-1. FIG. 1B. Human PD-1.

FIG. 2. CFSE assay to evaluate the functional effect of anti-PD1antibodies on the proliferation of HIV specific CD8 T cells.

FIG. 3. Concentration response binding of anti-PD-1 antibodies to cellsurface PD-1 on activated CD4 T-cells.

FIG. 4. Antibody classes.

FIG. 5. Proliferation relative to controls (NEG and Peptide 8). FIG. 5A.Class 1 mAb 137F2: strongly competitive (IC50˜200 pM; Imax=100%). FIG.5B. Class 2 mAb 139D6: partially competitive (IC50˜300 pM; Imax=50%).FIG. 5C. Class 2 mAb 136B4: non-competitive. FIG. 5D. Class 1 mAb 137F2:strongly competitive. FIG. 5E. Class 1 mAb 132F7: partially competitive(IC50˜200 pM; Imax=100%). FIG. 5F. Class 1 mAb 136B4: non-competitive.

FIG. 6. Restoration of HIV peptide specific CD8 T-cell proliferationmediated by anti-PD-1 antibodies binding to different epitopes in afunctional exhaustion recovery assay. FIG. 6A. HIV peptide 8 +BMS-5C4,MK3475, 135H12, 122H2, 139D6, 135E10, or 136B5. FIG. 6B. HIV peptide 8+MK3475, 135H12, 122H2, 137F2, 135C12, 135D11, or 136B5.

FIG. 7. Enhanced restoration of HIV peptide specific CD8 T-cellproliferation mediated by the combination of anti-PD-1 antibodiesbinding to different PD-1 epitopes in a functional exhaustion recoveryassay.

FIG. 8. Synergy between a first binding agent that blocks theinteraction of PD-1 and PD-L1 with a second binding agent that does notblock the interaction of PD-1 and PD-L1.

SUMMARY OF THE DISCLOSURE

This disclosure relates to binding agents with specificity forprogrammed cell death 1 (PD-1) (e.g., human PD-1) and to methods forusing the same such as to treat, prevent and/or ameliorate infection(e.g., by human immunodeficiency virus (HIV)), cancer and/or anautoimmune condition. Functional assays for identifying binding agentsthat interact with PD-1 are also provided. Combinations of bindingagents, such as a first binding agent that blocks the interaction ofPD-1 and PD-L1 with a second binding agent that does not block theinteraction of PD-1 and PD-L1, are also provided that actsynergistically to rescue T cells from exhaustion.

DETAILED DESCRIPTION

This disclosure relates to binding agents that bind programmed celldeath (PD-1) protein (e.g., SEQ ID NO:1, FIG. 1A, FIG. 1B of U.S. Pat.No. 5,698,520 (Honjo, et al.) which is hereby incorporated by referencein its entirety) (e.g., human PD-1) on the surface of cells in vitroand/or in vivo. The binding agents may also bind isolated PD-1polypeptide (e.g., human PD-1) and/or fragments and/or derivativesthereof, typically in vitro. Also provided are methods for using suchbinding agents to diagnose, treat, prevent and/or ameliorate one or morediseases associated with the existence of cells expressing PD-1. Forinstance, the binding agents may be antibodies (e.g., monoclonalantibodies) that may react with and/or bind to the epitopes of PD-1. The“binding agents” described herein may include, for example, an agonistor an antagonist of PD-1. An agonist binding agent is one that is nottypically capable of restoring T-cell function and/or expression ofPD-1. An agonist PD-1 binding agent may be useful for treatingautoimmune diseases and others in which PD-1 expressing cells areinvolved in disease progression. In contrast, an antagonist bindingagent is one capable for restoring T-cell function and/or expression ofPD-1. For instance, a PD-1 antagonist binding agent may be capable ofrestoring the function of PD-1 expressing T-cells from functionalexhaustion as is known to occur in HIV infection and in a variety oftumors. Restoration of T cell function may be determined by, forinstance, measuring proliferation, cytokine production, cytotoxicactivity or other characteristics of such cells. Another use for thebinding agents described herein is the selective targeting andelimination of HIV-infected CD4 T-cell populations containingreplication competent HIV (e.g., in a latent and/or replication state).Such PD-1 expressing cells expressing PD-1 are known to serve as a majorcell reservoir for replication competent HIV. A potential mechanism forthe elimination of these CD4 T-cell populations is antibody-dependentcellular cytotoxicity (ADCC) using the binding agents described herein(e.g., mono- and/or bi-specific PD-1 antibodies). In some embodiments,one or more PD-1 antagonistic binding agents having, for instance,different specificities (e.g., recognizing different epitopes) may becombined to induce rescue of antigen-specific CD8 T-cells fromfunctional exhaustion caused by PD-1 expression in those cells (e.g.,restoring or improving proliferation, cytokine production and/orcytotoxic activity). In some embodiments, the binding agents describedherein may also provide for the selective elimination and/or suppressionof PD-1 expressing cells. In some embodiments, the PD-1 agonist bindingagents described herein may be used to supress and/or eliminate PD-1expressing cells to treat, for instance, infectious diseases (e.g.,HIV), cancer, and/or, especially, autoimmune conditions. Otherembodiments, uses and the like are described below.

The binding agents may be antibodies such as monoclonal antibodies thatmay comprise, for instance, any one or more of the amino acid sequencesshown in Table 1 (and/or one or more fragments and/or derivativesthereof). This disclosure also provides for the use of such monoclonalantibodies to isolate, identify, and/or target cells expressing PD-1. Incertain embodiments, these monoclonal antibodies may be reactive againstPD-1 expressed on the surface of cells. The term “antibody” or“antibodies” may refer to whole or fragmented antibodies in unpurifiedor partially purified form (e.g., hybridoma supernatant, ascites,polyclonal antisera) or in purified form. The antibodies may be of anysuitable origin or form including, for example, murine (e.g., producedby murine hybridoma cells), or expressed as humanized antibodies,chimeric antibodies, human antibodies, and the like. For instance,antibodies may be wholly or partially dervied from human (e.g., IgG(IgG1, IgG2, IgG2a, Ig2b, IgG3, IgG4), IgM, IgA (IgA1 and IgA2), IgD,and IgE), canine (e.g., IgGA, IgGB, IgGC, IgGD), chicken (e.g., IgA,IgD, IgE, IgG, IgM, IgY), goat (e.g., IgG), mouse (e.g., IgG, IgD, IgE,IgG, IgM), and/or pig (e.g., IgG, IgD, IgE, IgG, IgM), rat (e.g., IgG,IgD, IgE, IgG, IgM) antibodies, for instance. Methods of preparing,utilizing and storing various types of antibodies are well-known tothose of skill in the art and would be suitable in practicing thepresent invention (see, for example, Harlow, et al. Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, 1988; Harlow, et al.Using Antibodies: A Laboratory Manual, Portable Protocol No. 1, 1998;Kohler and Milstein, Nature, 256:495 (1975)); Jones et al. Nature,321:522-525 (1986); Riechmann et al. Nature, 332:323-329 (1988); Presta(Curr. Op. Struct. Biol., 2:593-596 (1992); Verhoeyen et al. (Science,239:1534-1536 (1988); Hoogenboom et al., J. Mol. Biol., 227:381 (1991);Marks et al., J. Mol. Biol., 222:581 (1991); Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner etal., J. Immunol., 147(1):86-95 (1991); Marks et al., Bio/Technology 10,779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison,Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14,845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonbergand Huszar, Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat.Nos. 4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;and, 5,661,016). In certain applications, the antibodies may becontained within hybridoma supernatant or ascites and utilized eitherdirectly as such or following concentration using standard techniques.In other applications, the antibodies may be further purified using, forexample, salt fractionation and ion exchange chromatography, or affinitychromatography using Protein A, Protein G, Protein A/G, and/or Protein Lligands covalently coupled to a solid support such as agarose beads, orcombinations of these techniques. The antibodies may be stored in anysuitable format, including as a frozen preparation (e.g., −20° C. or−70° C.), in lyophilized form, or under normal refrigeration conditions(e.g., 4° C.). When stored in liquid form, for instance, it is preferredthat a suitable buffer such as Tris-buffered saline (TBS) or phosphatebuffered saline (PBS) is utilized. In some embodiments, the bindingagent may be prepared as an injectable preparation, such as insuspension in a non-toxic parenterally acceptable diluent or solvent.Suitable vehicles and solvents that may be utilized include water,Ringer's solution, and isotonic sodium chloride solution, TBS and/orPBS, among others. Such preparations may be suitable for use in vitro orin vivo may be prepared as is known in the art and the exact preparationmay depend on the particular application.

However, the binding agents described are not in any way limited toantibodies. For example, the binding agent may be any compoundexhibiting similar binding properties as another (e.g., a mimetic). Forexample, an exemplary binding agent may be one that binds PD-1 and/orcan compete with binding agent having specificity therefor (e.g., amonoclonal antibody). In some embodiments, the mimetic may exhibitsubstantially the same affinity in binding assays as the binding agent(e.g., monoclonal antibody) to which it is being compared. The affinitya particular binding agent may be measured by any suitable assayincluding but not limited to FACS staining of endogenous cell surfacePD-1 on activated CD4 T cells as described in the Examples. One bindingagent may be said to have “substantially the same affinity” as anotherwhere the measurements (e.g., nm) are within about any of1-20,1-5,5-10,10-15, or 15-20 percent of one another. Exemplary mimeticsmay include, for example, organic compounds that specifically bind PD-1,or an affibody (Nygren, et al. FEBS J. 275 (11): 2668-76 (2008)),affilin (Ebersbach, et al. J. Mol. Biol. 372 (1): 172-85 (2007)),affitin (Krehenbrink, et al. J. Mol. Biol. 383 (5): 1058-68 (2008)),anticalin (Skerra, A. FEBS J. 275 (11): 2677-83 (2008)), avimer(Silverman, et al. Nat. Biotechnol. 23 (12): 1556-61 (2005)), DARPin(Stumpp, et al. Drug Discov. Today 13 (15-16): 695-701 (2008)), Fynomer(Grabulovski, et al. J. Biol. Chem. 282 (5): 3196-3204 (2007)), Kunitzdomain peptide (Nixon, et al. Curr. Opin. Drug Discov. Devel. 9 (2):261-8 (2006)), and/or a monobody (Koide, et al. Methods Mol. Biol. 352:95-109 (2007)). Other mimetics may include, for example, a derivative ofan antibody (of, for example, the monoclonal antibody 1E4,1G10, and/or1G1) such as, for example, an F_(ab), F_(ab2), Fab' single chainantibody, F_(v), single domain antibody, mono-specific antibody,bi-specific antibody, tri-specific antibody, multi-valent antibody,chimeric antibody, canine-human chimeric antibody, canine-mouse chimericantibody, antibody comprising a canine Fc, humanized antibody, humanantibody, caninized, CDR-grafted antibody, shark antibody, nanobody,canelid antibody, microbody, and/or intrabody, or derivative thereof.Other binding agents are also provided herein as would be understood byone of ordinary skill in the art.

Any method known to those of ordinary skill in the art may be used togenerate binding agents having specificity for (e.g., binding to) PD-1.For instance, to generate and isolate monoclonal antibodies an animalsuch as a mouse may be administered (e.g., immunized) with one or morePD-1 proteins (e.g., PD-1 Fc fusion protein and/or PD-1 His tagprotein). Animals exhibiting serum reactivity to PD-1 expressed onactivated human T lymphocytes (as determined by, for instance, flowcytometry and/or microscopy) may then be selected for generation ofanti-PD-1 hybridoma cell lines. This may be repeated for multiplerounds. For instance, the primary criteria for the first round ofbinding agent selection may be include but are not limited to: i) levelof staining of PD-1 on activated human T lymphocytes by flow cytometry;(ii) diversity of CDR VH and VL sequences as compared to those of theexisting anti-PD-1 antibodies; and, (iii) epitope mapping performed bycompetitive binding studies with PD-1 conjugated Luminex beadspre-coupled with PD-L1 or one of several commercially availableanti-PD-1 antibodies binding to different epitopes on PD-1. An exemplaryfirst or second round of selection may also include, for instance,affinity binding (not a primary criteria since it may not correlate withthe stimulatory potential of anti-PD-1 antibodies); and/or, functionalcharacterization to identify the binding agent as an agonist or anantagonist.

As described in Example 1 herein, for instance, the ExhaustionFunctional Recovery Assay (EFRA) may be used. In this assay, testbinding agents may be assayed for the ability to rescue immune cellssuch as T cells from exhaustion. This may be determined by measuring theability of a binding agent to restore proliferation to such cells in thepresence of an antigen, such as a test peptide derived from a virus suchas human immunodeficiency virus (HIV). Proliferation is measured in aCFSE assay in comparison to a control, such as the test peptide alone ora positive control anti-PD-1 antibody such as MK-3475 (pembrolizumab).In some embodiments, a binding agent is determined to restoreproliferation where the comparison shows a significant difference (suchas a P value of <0.001) compared to either a peptide alone control orpeptide with an isotype control mouse IgG1 antibody. This assay may beused to identify binding agents (such as antibodies) that compete withother binding agents for binding to PD-1 (such as PD-L1 or PD-L2) and/orlead to the functional restoration of immune cells. Example 1 alsodescribes two methods of epitope mapping the antibodies listed in Table2 using Luminex-based assays. In one biochemical assay, a PD-1 Fc fusionprotein is bound to beads and competitive binding studies are performedbetween the anti-PD-1 antibodies described in Table 2 and one of twodifferent commercially available anti-PD-1 antibodies. Example 1describes four classes of monoclonal antibodies binding to distinctepitopes on PD-1 that were: class 1 (competitive with a first monoclonalantibody that blocks the interaction of PD-1 with PD-L1), class 2(competitive with a second monoclonal antibody that binds PD-1 but doesnot block the interaction of PD-1 with PD-L1), class 3 (competitive withboth the first and second monoclonal antibodies), and class 4(non-competitive with either the first or second antibodies). In aseparate assay, competition for binding to a recombinant PD-1 proteinwas evaluated for the anti-PD-1 antibodies listed in Table 2 and abiotinylated PD-L1 recombinant protein. Antibodies that inducedproliferation in the EFRA were identified from all four binding classesthat are proposed of binding to different epitopes on PD-1. Likewise,the EFRA allowed for the identification of anti-PD-1 antibodies thatwere either competitive, partially competitive or non-competitive withthe PD-1/PD-L1 interaction and specifically restored proliferativefunction to HIV specific CD8 T-cell.

Combinations of binding agents may also be identified, such as thosedescribed in Example 2. In some embodiments, the combinations may beidentified to provide statistically significant differences from resultsobtained using only one or more of the binding agents and not others. Insome embodiments, combinations exhibiting synergistic ability to restoreimmune cell function may be identified. In some embodiments, thecombination may comprise a first binding agent that blocks theinteraction of PD-1 and PD-L1 with a second binding agent that does notblock the interaction of PD-1 and PD-L1. The first and second bindingagents may be different entities such as two or more differentmonoclonal antibodies or derivatives thereof, or may be found on thesame entity such as a bi-functional antibody (a single antibody orderivative thereof comprising multiple binding specificities). Forinstance, an exemplary bi-functional antibody may comprise a firstbinding region that blocks the interaction of PD-1 and PD-L1 and asecond binding region that does not block the interaction of PD-1 andPD-L1. Also contemplated are combinations that provide multiple types ofeach binding agent. For instance, the combination may comprise multipletypes of binding agents that block the interaction of PD-1 and PD-L1with one or more that does not block the interaction of PD-1 and PD-L1.In some embodiments, the combination may comprise one or more of bindingagents that block the interaction of PD-1 and PD-L1 with multiplebinding agents that do not block the interaction of PD-1 and PD-L1. Insome embodiments, the combination may comprise multiple binding agentsthat block the interaction of PD-1 and PD-L1 with multiple bindingagents that do not block the interaction of PD-1 and PD-L1. Suchcombinations as described herein may also be combined with one or moreother agents that may effect immune cell function such as antibodiesagainst CTLA-4 and the like. One of ordinary skill in the art wouldrecognize that many such combinations may be suitable for use asdescribed herein.

Where the binding agent is an antibody, it may be identified withreference to the nucleotide and/or amino acid sequence corresponding tothe variability and/or complementarity determining regions (“CDRs”)thereof. The variable region/CDR sequences may be used in combinationwith one or more other variable region/CDR amino acid sequences. Thevariable region/CDR amino acid sequences may alternatively and/or alsobe adjoined to one or more types of constant region polypeptides of anantibody molecule. For instance, the CDR amino acid sequences shown inTables 1A and 1B may be adjoined to or associated with the constantregions of any antibody molecule of the same or a different species(e.g., human, goat, rat, sheep, chicken) and/or antibody subtype of thatfrom which the CDR amino acid sequence was derived. For instance, anexemplary binding agent may be, or may be derived from, or may berelated to the monoclonal antibody produced by the hybridomas listed in,and/or may have about the same affinity and/or proliferation effect,and/or exhibit the same binding class shown in Table 2, and/or may haveany one or more of the amino acid sequences of SEQ ID NOS. 1-138 and/oras shown in Tables 1A and 1B. The binding agent may comprise an antibodyheavy and/or a light chain that each comprises one or more constantand/or variable regions. The variable regions typically comprise one ormore CDRs that may determine the binding specificity of the antibody.The monoclonal antibodies may also be identified by analysis of theamino acid sequences of (e.g., which may be encoded by such nucleotidesequences) such variable regions. For instance, exemplary amino acidsequences of the heavy chain CDRs of binding agents that bind PD-1 mayinclude any one or more of comprising at least one amino acid sequenceselected from the group consisting of SEQ ID NOS. 1-138, and/or anyother shown in Tables 1A and/or 1B. Any of the amino acid sequencesdescribed herein, and/or any fragments and/or derivatives thereof mayalso be combined with any other variable region and/or CDR in any orderand/or combination to form hybrid and/or fusion binding agents and/orinserted into other heavy and/or light chain variable regions usingstandard techniques. Exemplary combinations of CDRs (e.g., combinationof heavy and/or light chain CDR1, CDR2 and CDR3 amino acid sequences)that may be found in a PD-1 (e.g., human PD-1) binding agent of thisdisclosure may include, for instance, the embodiments shown in Tables 1Aand/or 1B.

TABLE 1A Heavy chain: Amino acids sequence Clone CDR1 CDR2 CDR3 122F10DDFLH RIDPANGESRYAPKFQD TDYRGYYYAMDY (SEQ ID NO: 1) (SEQ ID NO: 24)(SEQ ID NO: 47) 139D6 NYYIH SIYPNYGDTNYNQKVKD GYSYAMDY (SEQ ID NO: 2)(SEQ ID NO: 25) (SEQ ID NO: 48) 135D1 NYYIH SIYPNYGETNYNQEFKG GYSYAMDY(SEQ ID NO: 3) (SEQ ID NO: 26) (SEQ ID NO: 49) 134D2 SNWMHAVNPGNSDTTYNQKFKG GRSYDGSFDY (SEQ ID NO: 4) (SEQ ID NO: 27)(SEQ ID NO: 50) 121G1 RYWMH NIDPSDSTTHYNPKFRD DLDDFYVGSHEDFDY(SEQ ID NO: 5) (SEQ ID NO: 28) (SEQ ID NO: 51) 136B5 SNWMHAVYPGNSDTTYNQNFKG GRSYDGSFDY (SEQ ID NO: 6) (SEQ ID NO: 29)(SEQ ID NO: 52) 127C2 NSYIH WISPGDGSTNYNEKFKG EEYDYDNY (SEQ ID NO: 7)(SEQ ID NO: 30) (SEQ ID NO: 53) 137F2 NYWIG DIYPGGGYTNYNEKFKG GYDFVLDR(SEQ ID NO: 8) (SEQ ID NO: 31) (SEQ ID NO: 54) 138H5 SYAMSTISGGGADTYYLDNVKG QRGENLFAH (SEQ ID NO: 9) (SEQ ID NO: 32)(SEQ ID NO: 55) 140A1 SDYAWN YINYSGYTNYNPFLKS YGGSYPWNFDV(SEQ ID NO: 10) (SEQ ID NO: 33) (SEQ ID NO: 56) 135H12 SYWINNIYPGSSSIDYNEKFKS GLYWYFDV (SEQ ID NO: 11) (SEQ ID NO: 34)(SEQ ID NO: 57) 131D11 SSYIH WIFPGDGKTNYNEKFRD NDFDRGVY (SEQ ID NO: 12)(SEQ ID NO: 35) (SEQ ID NO: 58) 132F7 NHGMS SINTGGYSTYYPDNVKG DDYNWFAY(SEQ ID NO: 13) (SEQ ID NO: 36) (SEQ ID NO: 59) 126E4 NYWIGDIYPGSEYENYNEKFKG GYDFVLDH (SEQ ID NO: 14) (SEQ ID NO: 37)(SEQ ID NO: 60) 135G1 DSYIH RIDPAHGNVIYASKFRD IYYDYGEGDF (SEQ ID NO: 15)(SEQ ID NO: 38) (SEQ ID NO: 61) 136E10 DTYIH RIDLANDDILYASKFQGIYYDYGEGDY (SEQ ID NO: 16) (SEQ ID NO: 39) (SEQ ID NO: 62) 135C12 NFYIHSIYPNYGDTAYNQKFKD GYSYAMDY (SEQ ID NO: 17) (SEQ ID NO: 40)(SEQ ID NO: 63) 136F4 DSYIH RIDPARDNIIYASKFRD IYYDYGEGDY (SEQ ID NO: 18)(SEQ ID NO: 41) (SEQ ID NO: 64) 136B4 DDFLH RIDPANGESRYAPQFQDTDYRGYYYAMDY (SEQ ID NO: 19) (SEQ ID NO: 42) (SEQ ID NO: 65) 135E10SYFMS GISTGGADTYYADSMKG LSHYYDGIPLDC (SEQ ID NO: 20) (SEQ ID NO: 43)(SEQ ID NO: 66) 140G5 NHGMS SISGGGDNTYYPDNLKG VRQLGLHRAAMDY(SEQ ID NO: 21) (SEQ ID NO: 44) (SEQ ID NO: 67) 122H2 NYWIGDIYPGGDHKNYNEKFKD GFDFVLDY (SEQ ID NO: 22) (SEQ ID NO: 45)(SEQ ID NO: 68) 139F11 SFAMS TITGGGVNTYYPDTVKG QAIYDGHYVLDY(SEQ ID NO: 23) (SEQ ID NO: 46) (SEQ ID NO: 69)

TABLE 1B Light chain: Amino acids sequence Clone CDR1 CDR2 CDR3 122F10KSSQSVLYSSNQKNYLA WASTRES HQYLSSYT (SEQ ID NO: 70) (SEQ ID NO: 93)(SEQ ID NO: 116) 139D6 SASQGISDGLN HTSTLHS QQYSKFPLT (SEQ ID NO: 71)(SEQ ID NO: 94) (SEQ ID NO: 117) 135D1 SASQGISNGLN HTSTLHS QQYSKFPLT(SEQ ID NO: 72) (SEQ ID NO: 95) (SEQ ID NO: 118) 134D2 KASQDINKYIAYTSTLRP LQYDNLWT (SEQ ID NO: 73) (SEQ ID NO: 96) (SEQ ID NO: 119) 121G1RSSQSIVYSNGNTYLE KVSHRFS FQGSHVPYT (SEQ ID NO: 74) (SEQ ID NO: 97)(SEQ ID NO: 120) 136B5 KASQDINKYMA YTSTLRP LQYDNLWT (SEQ ID NO: 75)(SEQ ID NO: 98) (SEQ ID NO: 121) 127C2 KASQNVGTNVG SASYRYN QQYNTYPWT(SEQ ID NO: 76) (SEQ ID NO: 99) (SEQ ID NO: 122) 137F2 KSSQSLFNSETQKNYLAWASTRES KQSYTLRT (SEQ ID NO: 77) (SEQ ID NO: 100) (SEQ ID NO: 123) 138H5LASQTIGTWLA AATSLAD QQLYSTPWT (SEQ ID NO: 78) (SEQ ID NO: 101)(SEQ ID NO: 124) 140A1 RSSQTIVHNNGDTYLE KISNRFF FQGSHVPYT(SEQ ID NO: 79) (SEQ ID NO: 102) (SEQ ID NO: 125) 135H12KSSQSLFNSGTRKNYLA WASTRDS KQSYNLYT (SEQ ID NO: 80) (SEQ ID NO: 103)(SEQ ID NO: 126) 131D11 KASQNVDTNVA SASYRYN QQYNNYPYT (SEQ ID NO: 81)(SEQ ID NO: 104) (SEQ ID NO: 127) 132F7 KSSQSLLNSGNQKNYLT WASTRESQSDYSYPLT (SEQ ID NO: 82) (SEQ ID NO: 105) (SEQ ID NO: 128) 126E4KSSQSLFNSGTRKSYLA WASTRET MQSYNLRT (SEQ ID NO: 83) (SEQ ID NO: 106)(SEQ ID NO: 129) 135G1 HASQNINVWLS KASNLHT QQGQSWPLT (SEQ ID NO: 84)(SEQ ID NO: 107) (SEQ ID NO: 130) 136E10 HASQNINVWLS KASNLHT QQGQSYPLT(SEQ ID NO: 85) (SEQ ID NO: 108) (SEQ ID NO: 131) 135C12 SASQGISGDLNHTSSLHS QYYSKDLLT (SEQ ID NO: 86) (SEQ ID NO: 109) (SEQ ID NO: 132)136F4 HASQNINVWLS KASNLHT QQGQSWPLT (SEQ ID NO: 87) (SEQ ID NO: 110)(SEQ ID NO: 133) 136B4 KSSQSVLYSSNQKNYLA WASTRES HQYLSSYT(SEQ ID NO: 88) (SEQ ID NO: 111) (SEQ ID NO: 134) 135E10 RASESVDNSGVSFLTAASNQGS QQTKEVPWT (SEQ ID NO: 89) (SEQ ID NO: 112) (SEQ ID NO: 135)140G5 KASQSVSDDVS SAFFRYP QQDYSSPLT (SEQ ID NO: 90) (SEQ ID NO: 113)(SEQ ID NO: 136) 122H2 KSSQSLFNSGTRKNYLA WASTRES MQSFNLRT(SEQ ID NO: 91) (SEQ ID NO: 114) (SEQ ID NO: 137) 139F11 RTSGNIHNYLANVKTLTD QQFWSIPWT (SEQ ID NO: 92) (SEQ ID NO: 115) (SEQ ID NO: 138)In addition, any of SEQ ID NOS. 1-69 may be combined with any one ormore of SEQ ID NOS. 70-138 into a binding agent. In preferredembodiments, the heavy chain CDRs of each clone are combined with theirrespective light chain CDRs into a binding agent. In some embodiments,the binding agent may comprise the heavy chain CDRs and light chain CDRsshown below:

-   -   122F10 (SEQ ID NOS. 1, 24, 47, 70, 93, and 116);    -   139D6 (SEQ ID NOS. 2, 25, 48, 71, 94, and 117);    -   135D1 (SEQ ID NOS. 3, 26, 49, 72, 95, and 118);    -   134D2 (SEQ ID NOS. 4, 27, 50, 73, 96, and 119);    -   121G1 (SEQ ID NOS. 5, 28, 51, 74, 97, and 120);    -   136B5 (SEQ ID NOS. 6, 29, 52, 75, 98, and 121);    -   127C2 (SEQ ID NOS. 7, 30, 53, 76, 99, and 122);    -   137F2 (SEQ ID NOS. 8, 31, 54, 77, 100, and 123);    -   138H5 (SEQ ID NOS. 9, 32, 55, 78, 101, and 124);    -   140A1 (SEQ ID NOS. 10, 33, 56, 79, 102, and 125);    -   135H12 (SEQ ID NOS. 11, 34, 57, 80, 103, and 126);    -   131D11 (SEQ ID NOS. 12, 35, 58, 81, 104, and 127);    -   132F7 (SEQ ID NOS. 13, 36, 59, 82, 105, and 128);    -   126E4 (SEQ ID NOS. 14, 37, 60, 83, 106, and 129);    -   135G1 (SEQ ID NOS. 15, 38, 61, 84, 107, and 130);    -   136E10 (SEQ ID NOS. 16, 39, 62, 85, 108, and 131);    -   135C12 (SEQ ID NOS. 17, 40, 63, 86, 109, and 132);    -   136F4 (SEQ ID NOS. 18, 41, 64, 87, 110, and 133);    -   136B4 (SEQ ID NOS. 19, 42, 65, 88, 111, and 134);    -   135E10 (SEQ ID NOS. 20, 43, 66, 89, 112, and 135);    -   140G5 (SEQ ID NOS. 21, 44, 67, 90, 113, and 136);    -   122H2 (SEQ ID NOS. 22, 45, 68, 91, 114, and 137); or    -   139F11 (SEQ ID NOS. 23, 46, 69, 92, 115, and 138).        Other combinations may also be useful as may ascertained by one        of ordinary skill in the art.

Binding agents comprising the CDRs of Tables 1A and/or 1B, or those ofthe immediately preceding paragraph, may also exhibit the followingcharacteristics:

TABLE 2 Antibody EFRA competition % relative with the to peptideAffinity* Binding PD-1/PD-L1 stimulation Clone (nM) Class**interaction*** alone^(‡) 122F10 2.2 4 Non-competitive 146% 139D6 2.4 2Partial competition 195% 135D1 6.5 2 Partial competition 187% 134D2 4.84 Competitive 205% 121G1 11.9 4 Non-competitive 120% 136B5 7.7 4Competitive 200% 127C2 1.0 2 Non-competitive 100% 137F2 1.5 1Competitive 250% 138H5 1.6 3 Competitive 210% 140A1 1.4 3 Competitive160% 135H12 1.9 1 Competitive 190% 131D11 2.7 1 Competitive 180% 132F7100 2 Non-competitive 210% 126E4 0.5 4 Competitive 130% 135G1 32 4 NA138% 136E10 7.1 4 Non-competitive 148% 135C12 1.7 2 Partial competition195% 136F4 8.3 4 Non-competitive 108% 136B4 1.4 2 Non-competitive 185%135E10 1.5 3 Competitive 165% 140G5 1.6 1 Competitive 205% 122H2 4.3 1Competitive 200% 139F11 3.1 1 Competitive 250% *Binding affinity for theantibodies listed in Table 1 was evaluated by FACS staining ofendogenous cell surface PD-1 on activated CD4 T cells. **Binding classwas determined by Luminex assay competitive binding studies. Bindingclass 1 mAb clones are competitive with the EH12.2H7 clone commercialantibody, class 2 mAb clones are competitive with the J116 clonecommercial antibody, class 3 mAb clones are competitive with bothEH12.2H7 and J116 antibodies and class 4 mAb clones bind in the presenceof both EH12.2H7 and J116 antibodies. ***Antibody competition with thePD-1/PD-L1 interaction was determined in a second Luminex binding assay.In this assays, PD-1 Fc fusion protein coated beads were incubated inthe absence or presence of an anti-PD-1 antibody from Table 2 at aconcentration of 20 nM. A fixed concentration of 1.25 nM biotinylatedPD-L1, approximately equivalent to the IC₅₀ of the PD-1/PD-L1interaction, was then incubated with the PD-1/antibody complex and PD-L1binding was detected by fluorescence with phycoerythrin labeledstreptavidin. Based on PD-L1 binding to the PD-1/antibody complex,antibodies were defined as being competitive, partially competitive ornon-competitive with the PD-1/PD-L1 interaction. ^(‡)Proliferativeeffect is evaluated using a CFSE assay (an embodiment of the ExhaustionFunctional Recovery Assay, “EFRA”). PBMCs isolated from a chronicallyinfected HIV subject were stimulated with an HIV specific peptide in thepresence and absence of an anti-PD-1 antibody. Following a 6 dayincubation, proliferation of HIV specific CD8 T cells was evaluated inthe anti-PD-1 treated samples relative to the peptide alone control. NA= not available

Binding affinity may be determined by any technique available to thoseof ordinary skill in the art. The binding affinity data presented inTable 2 was evaluated by flow cytometry staining of endogenous cellsurface PD-1 on CD4 T cells that were stimulated for a period of 3 to 6days with phytohaemagglutinin (PHA). Binding class may also bedetermined by any technique available to those of ordinary skill in theart. The binding class data presented in Table 2 was determined byLuminex assay competitive binding studies. In Table 2, binding class 1mAb antibodies are those determined to be competitive with the EH12.2H7clone commercial antibody (available from BioLegend, San Diego,Calif.(e.g., Cat. No. 329905)); class 2 antibodies are those determinedto be competitive with the J116 clone commercial antibody (availablefrom Affymetrix eBioscience, San Diego, Calif. (e.g., Cat. No.16-9989-80)); and class 3 antibodies are those determined to becompetitive with both EH12.2H7 and J116 antibodies; and class 4 mAbclone antibodies are those determined to bind PD-1 in the presence ofboth EH12.2H7 and J116 antibodies.

Proliferative effect may be determined by any technique available tothose of ordinary skill in the art. For instance, the EFRA systemdescribed above and used in Example 1 may be used. Such an assay wasused to determine the proliferative effect data presented in Table 2.Briefly, a carboxyfluorescein succinimidyl ester (CFSE) assay in whichperipheral blood mononuclear cells (PBMCs) were isolated from achronically infected HIV subject and stimulated with an HIV-specificpeptide in the presence and absence of an anti-PD-1 antibody. A controlanti-PD1 antibody (the Merck antibody MK-3475) was also tested as apositive control. Following a six-day incubation, proliferation ofHIV-specific CD8 T cells was evaluated in the anti-PD-1 treated samplesrelative to the peptide alone control and the result expressed as apercentage above control (“Proliferation effect”).

In some embodiments, the techniques used to identify and characterizePD-1 binding agents such as antibodies may be combined to provide asystem for identifying and characterizing such binding agents. Forinstance, one or more candidate binding agents such one or moremonoclonal antibodies may be assayed by EFRA or a similar assay todetermine the ability of the candidate binding agent to restore functionto immune cells as measured by, for instance, proliferation in thepresence of an immunogenic peptide. In some embodiments, this type ofassay may be used as an initial screen to ensure the candidate bindingagents to be further studied are capable of restoring immune cellfunction. In some embodiments, these types of assays may be followed byone for determining the binding affinity to immune cells such asactivated peripheral blood mononuclear cells (PBMCs). In someembodiments, this assay may use a technique such as fluorescenceactivated cell sorting (FACS). In some embodiments, the assay mayinclude the presence or absence of non-specific binding and/orcompetitive binding studies using known binding reagents such asanti-PD1 antibody (e.g., the Merck antibody MK-3475, also know aspembrolizumab). These assays may then be followed by sequencing of theCDRs of the candidate binding agents such as provided in Tables 1Aand/or 1B above. Together, then, the EFRA, affinity determination,epitope mapping studies and CDR identification methods described hereinprovide a system with which a candidate binding agent may be identified.

Any of the amino acid sequences of Tables 1A and/or 1B (and/or any oneor more fragments and/or derivatives thereof) may be also substituted byany other amino acid as desired by one of ordinary skill in the art. Forexample, one of skill in the art may make conservative substitutions byreplacing particular amino acids with others as shown in Table 3 below.The specific amino acid substitution selected may depend on the locationof the site selected. Such conservative amino acid substitutions mayinvolve a substitution of a native amino acid residue with a non-nativeresidue such that there is little or no effect on the size, polarity,charge, hydrophobicity, or hydrophilicity of the amino acid residue atthat position and, in particular, does not result in decreased PD-1binding.

TABLE 3 Exemplary Preferred Original Conservative ConservativeSub- AminoAcid Substitutions of the stitution of the Residues Original AminoOriginal Amino in SEQ ID Acid Residues of Acid Residues of NOS. 1-138SEQ ID NOS. 1-138 SEQ ID NOS. 1-138 Ala Val, Leu, Ile Val Arg Lys, Gln,Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp AspGly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe,Norleucine Leu Leu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4Diamino-butyric Acid, Gln, Arg Asn Met Leu, Phe, Ile Leu Phe Leu, Val,Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr,Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala,Norleucine Leu

In some embodiments, this disclosure provides binding agents withmultiple specificities such that PD-1 and at least one other secondaryantigen (e.g., a cell surface protein) may be bound by a single bindingagent. In some embodiments, the secondary antigen may be one expressedby cells infected by an infectious agent. For instance, an exemplarysecondary antigen may be HIV Env antigen. Such binding agents may bindthe secondary antigen and/or may serve to neutralize the infectiousagent. In certain embodiments, such as for a bi-specific binding agenthaving dual specificity for PD-1 and an HIV antigen such as env and/oranother antigen, for instance. The HIV immunogen may be derived from anyof the subtypes described herein, or any other. In some embodiments,such binding agents may include: PD-1 agonist/Env binding; PD-1 agonistPD-1/Env binding and neutralization; PD-1 antagonist/Env binding; and/orPD-1 antagonist/PD-1/Env binding and neutralization. Given theprevalence of the various subtypes, it may be preferable to selectantigens from HIV-1 subtypes B and/or C. It may also be desirable toinclude binding agents having specificity for antigens from multiple HIVsubtypes (e.g., HIV-1 subtypes B and C, HIV-2 subtypes A and B, or acombination of HIV-1 and HIV-2 subtypes) in a single composition. Fortreating a disease such as cancer, it may be beneficial to obtainbinding agents with multiple PD-1 specificities (e.g., bi-specificPD-1a/PD1b antagonist PD-1 antibodies specific to two differentepitopes) and/or specificity to both PD-1 and one or more tumor antigens(e.g., cancer-testis (CT) antigen (i.e., MAGE, NY-ESO-1); melanocytedifferentiation antigen (i.e., Melan A/MART-1, tyrosinase, gp100);mutational antigen (i.e., MUM-1, p53, CDK-4); overexpressed ‘self’antigen (i.e., HER-2/neu, p53); and/or viral antigens (i.e., HPV, EBV)).The binding agents (e.g., monoclonal antibodies) may be generated asgenerally described above. The specificities of such binding agents maybe recombined into a single binding agent using techniques that arewidely available to those of ordinary skill in the art. In someembodiments, multiple single specifity binding agents may also becombined and used (e.g., administered) to provide an effective multiplespecificity reagent.

In some embodiments, the binding agents described herein may beconjugated to active agents to target and inhibit the function of and/or eliminate cell populations expressing PD-1 (and/or another antigenin the case of binding agents with multiple specificities). Forinstance, CD4⁺ T-cell populations containing replication competent HIVmay be targeted and eliminated using binding agent/drug conjugates(e.g., antibody-drug conjugates (ADC)). Mono- and/or bi-specificcandidate binding agents may be conjugated with one or more types ofdrugs (e.g., drugs damaging DNA, targeting microtubules). The bindingagents described herein and/or derivatives thereof may also be adjoinedto and/or conjugated to functional agents for in vitro and/or in vivouse. For instance, the binding agent may be adjoined to and/orconjugated to functional moieties such as cytotoxic drugs or toxins,and/or active fragments thereof such as diphtheria A chain, exotoxin Achain, ricin A chain, abrin A chain, curcin, crotin, phenomycin,enomycin, among others. Suitable functional moieties may also includeradiochemicals. Binding agents, such as antibodies, may be adjoined toand/or conjugated to the one or more functional agents using standardtechniques in the art.

In some embodiments, the binding agents may be administered inconjunction with other agents such as anti-infective agents (e.g.,antibiotics, anti-viral medications). For instance, the binding agentsdescribed herein may be combined with monoclonal antibodies and/or otherreagents such as Nivolumab (also known as MDX-1106, BMS-936558(Topalian, et al. N. Eng. J. Med. 2012; 366(26): 2443-2454), MDX-1106,ONO-4538, a fully human IgG4 mAb available from Bristol-Myers Squibb),Lambrolizumab (also known as MK-3475 and SCH 900475, a humanized IgG4monoclonal antibody available from Merck), Pidilizumab (a humanized IgG1monoclonal antibody available from CureTech), AMP-224 (a B7-DC/IgG1fusion protein available from GlaxoSmithKline/Amplimmune), and/or anantibody or other reagent or method described in any of U.S. Pat. No.8,354,509B2 (Carven, et al), U.S. Pat. No. 8,008,449B2 (Korman, et al),WO 2012/135408A1 (Manoj, et al.), US 2010/026617 (Carven, et al.), WO2011/110621A1 (Tyson, et al), U.S. Pat. No. 7,488,802B2 (Collins, etal.), WO 2010/029435A1 (Simon, et al.), WO 2010/089411A2 (Olive, D.), WO2012/145493A1 (Langermann, et al.), WO 2013/0435569A1 (Rolland, et al.),WO 2011/159877A2 (Kuchroo, et al.), U.S. Pat. No. 7,563,869B2 (OnoPharm.), U.S. Pat. No. 7,858,746B2 (Honjo, et al.), U.S. Pat. No.8,728,474B2 (Ono Pharm.), U.S. Pat. No. 9,067,999 (Ono Pharm.), and/orU.S. Pat. No. 9,067,999, each of which is hereby incorporated in itsentirety into this disclosure.

As mentioned above, the PD-1 binding agents described herein (e.g., aPD-1 antagonist) may be used to treat and/or prevent and/or amelioratethe symptoms of infection by HIV. As is well-known in the art, HIVisolates are now classified into discrete genetic subtypes. HIV-1 isknown to comprise at least ten subtypes (A1, A2, A3, A4, B, C, D, E, F1,F2, G, H, J and K) (Taylor et al, NEJM, 359(18):1965-1966 (2008)). HIV-2is known to include at least five subtypes (A, B, C, D, and E). SubtypeB has been associated with the HIV epidemic in homosexual men andintravenous drug users worldwide. Most HIV-1 immunogens, laboratoryadapted isolates, reagents and mapped epitopes belong to subtype B. Insub-Saharan Africa, India and China, areas where the incidence of newHIV infections is high, HIV-1 subtype B accounts for only a smallminority of infections, and subtype HIV-1 C appears to be the mostcommon infecting subtype. Any of these types of isolates may beaddressed using the binding agents described herein. One or more bindingagents may also be administered with or in conjunction with one or moreagents used to prevent, treat and/or ameliorate HIV such as for example,a protease inhibitor, an HIV entry inhibitor, a reverse transcriptaseinhibitor, and/or an anti-retroviral nucleoside analog. Suitablecompounds include, for example, Agenerase (amprenavir), Combivir(Retrovir/Epivir), Crixivan (indinavir), Emtriva (emtricitabine), Epivir(3tc/lamivudine), Epzicom, Fortovase/Invirase (saquinavir), Fuzeon(enfuvirtide), Hivid (ddc/zalcitabine), Kaletra (lopinavir), Lexiva(Fosamprenavir), Norvir (ritonavir), Rescriptor (delavirdine),Retrovir/AZT (zidovudine), Reyatax (atazanavir, BMS-232632), Sustiva(efavirenz), Trizivir (abacavir/zidovudine/lamivudine), Truvada(Emtricitabine/Tenofovir DF), Videx (ddl/didanosine), Videx EC (ddl,didanosine), Viracept (nevirapine), Viread (tenofovir disoproxilfumarate), Zerit (d4T/stavudine), and Ziagen (abacavir) may be utilized.Other suitable agents are known to those of skill in the art and may besuitable for use as described herein. Such agents may either be usedprior to, during, or after administration of the binding agents and/oruse of the methods described herein.

As mentioned above, the PD-1 binding agents described herein (e.g., aPD-1 antagonist) may be used to treat and/or prevent and/or amelioratethe symptoms of cancer. Exemplary cancers may include, for instance, anyof the breast, blood, colon, stomach, rectum, skeletal tissue, skin(e.g., melanoma) brain, lung, bladder, kidney, ovary, and/or liver,among others. One or more of the binding agents may also be combinedwith and/or administered with or in conjunction with one or more agentsused to prevent, treat and/or ameliorate cancer such as for example, analkylating agent (e.g., any nitrogen mustard, nitrosourea, tetrazine,aziridine, cisplatin and/or derivative thereof), anti-metabolite (e.g.,any of the methotrexates, pemetrexeds, fluoropyrimidines and/orderivative thereof), anti-microbtubule agent (e.g., vinca alkyloids,taxanes, podophyllotoxin and/or derivative thereof), topoisomerase Iand/or II inhibitors (e.g., a camptothecin, irinotecan, topotecan,etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone,aclarubicin and/or derivative thereof) and/or cytotoxic antibiotic(e.g., any anthracyclines, actinomycin, bleomycin, plicamycin, andmitomycin and/or derivative thereof). The one or more binding agents mayalso, or alternatively, be combined with one or more other bindingagents available to those of ordinary skill in the art for treating,preventing and/or ameliorating cancer such as, for example, Nivolumab,Lambrolizumab, Pidilizumab and/or other similar agents and/orderivatives thereof. Other suitable agents are known to those of skillin the art and may be suitable for use as described herein. Such agentsmay either be used prior to, during, or after administration of thebinding agents and/or use of the methods described herein.

As mentioned above, the PD-1 binding agents described herein (e.g., aPD-1 agonist) may be used to treat and/or prevent and/or ameliorate thesymptoms of autoimmunity. Exemplary autoimmune conditions may include,for instance, any in which PD-1 is involved in maintainingself-tolerance and/or one involving inflammatory T cells (e.g.,autoreactive or self antigen-specific T cells) such as, for instance,systemic lupus erythematosus (SLE), type I diabetes, rheumatoidarthritis, glomerulonephritis, and multiple sclerosis. Such PD-1 bindingagents may also be combined with other agents such as anti-CTLA-4 agents(e.g., ipilimumab). One or more of the binding agents may also becombined with and/or administered with or in conjunction with one ormore agents used to prevent, treat and/or ameliorate autoimmunity suchas, for example, glucocorticoids, cytostatics (e.g., alkylating agent,anti-metabolite, methotrexate, azathioprine, mercaptopurine, cytotoxicantibiotics (e.g., dactinomycin, anthracyclines, mitomycin C, bleomycin,mithramycin), antibodies (e.g., Atgam, Thymoglobuline, Simulect,Zenapax), drugs acting on immunophilins (e.g., ciclosporin, tacrolimus,sirolimus), interferons, opioids, TNF-binding agents (e.g., Remicade,Enbrel, Humira), mycophenolate, fingolimod, myriocin, and/or derivativesthereof. Other suitable agents are known to those of skill in the artand may be suitable for use as described herein. Such agents may eitherbe used prior to, during, or after administration of the binding agentsand/or use of the methods described herein.

In some embodiments, the binding agents may be adjoined to and/orconjugated to one or more detectable labels. For instance, suitabledetectable labels may include, for instance, fluorosceins (e.g.,DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT(Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 6-JOE;6-carboxyfluorescein (6-FAM); FITC;6-carboxy-1,4-dichloro-2′,7′-dichlorofluorescein (TET);6-carboxy-1,4-dichloro-2′, 4′, 5′, 7′-tetra-chlorofluorescein (HEX);6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein (JOE); Alexafluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594,610, 633, 635, 647, 660, 680, 700, 750); BODIPY fluorophores (e.g.,492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568, 564/570,576/589, 581/591, 630/650-X, 650/665-X, 665/676, FL, FL ATP,FI-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-XSE)), rhodamines (e.g., 110, 123, B, B 200, BB, BG, B extra,5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G,Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red,Rhod-2, ROX (6-carboxy-X-rhodamine), 5-ROX (carboxy-X-rhodamine),Sulphorhodamine B can C, Sulphorhodamine G Extra, TAMRA(6-carboxytetramethyl-rhodamine), Tetramethylrhodamine (TRITC), WT),Texas Red, and/or Texas Red-X. Other detectable labels known in the artmay also be suitable for use. Binding agents, such as antibodies, may beadjoined to and/or conjugated to the one or more detectable labels usingstandard techniques in the art.

In certain embodiments, a nucleic acid molecule encoding one or morebinding agents described herein may be inserted into one or moreexpression vectors, as discussed below in greater detail. In suchembodiments, the binding agent may be encoded by nucleotidescorresponding to the amino acid sequence. The particular combinations ofnucleotides (codons) that encode the various amino acids (AA) are wellknown in the art, as described in various references used by thoseskilled in the art (e.g., Lewin, B. Genes V, Oxford University Press,1994). The nucleotide sequences encoding the amino acids of said bindingagents may be ascertained with reference to Table 4, for example.Nucleic acid variants may use any combination of nucleotides that encodethe binding agent.

TABLE 4 Codons Encoding Amino Acids (AA) of SEQ ID NOS. 1-138 ofVariants Thereof Co- Co- Co- Co- AA don AA dons AA dons AA dons Phe (F)TTT Ser (S) TCT Tyr (Y) TAT Cys (C) TGT TTC TCC TAC TGC Leu (L) TTA TCATERM TAA TERM TGA TTG TCG TAG Trp (W) TGG CTT Pro (P) CCT His (H) CATArg (R) CGT CTC CCC CAC CGC CTA CCA Gln (Q) CAA CGA CTG CCG CAG CGG Ile(I) ATT Thr (T) ACT Asn (N) AAT Ser (S) AGT ATC ACC AAC AGC ATA ACA Lys(K) AAA Arg (R) AGA Met (M) ATG ACG AAG AGG Val (V) GTT Ala (A) GCT Asp(D) GAT Gly (G) GGT GTC GCC GAC GGC GTA GCA Glu (E) GAA GGA GTG GCG GAGGGGThose of ordinary skill in the art understand that the nucleotidesequence encoding a particular amino acid sequence may be easily derivedfrom the amino acid sequence and the information presented in Table 4.For instance, it may be deduced from the amino acid sequence DDFLH (SEQID NO.: 1) and the information presented in Table 4 that the amino acidsequence may be encoded by the nucleotide sequence GAT GAT TTT TTA CAT(SEQ ID NO.:139). Those of ordinary skill in the art would understandthat nucleotide sequences encoding SEQ ID NOS. 2-138 may be deduced inthe same way, and such nucleotide sequences are contemplated herein.Where the binding agents are antibodies, nucleotide sequences encodingthe variable regions thereof may also be isolated from the phage and/orhybridoma cells expressing the same cloned into expression vectors toproduce certain preparations (e.g., humanized antibodies). Methods forproducing such preparations are well-known in the art.

To determine the amino acid sequences of the variable regions (e.g.,CDRs) of interest, hybridoma cells from mice immunized with a PD-1antigen/immunogen may be selected using the functional assays describedherein and cloning techniques that are readily available to those ofordinary skill in the art. For instance, to isolate and sequence nucleicacids encoding the heavy and light chain variable regions of theselected hybridomas, total RNA may be extracted from fresh hybridomacells using TRIzol reagent according to the manufacturer's protocol.cDNA may be synthesized from the RNA using isotype-specific anti-senseprimers or universal primers using standard techniques (e.g., followingthe technical manual of PrimeScript™ 1st Strand cDNA Synthesis Kit).Polymerase chain reaction (PCR) may then be performed to amplify thenucleic acids encoding the variable regions (heavy and light chains) ofthe antibody produced by the selected hybridoma, which may then becloned into a standard cloning vector separately and sequenced. ColonyPCR screening may then be performed to identify clones with inserts ofcorrect sizes. Preferably, no less than five single colonies withinserts of correct sizes are sequenced for each antibody variableregion. Standard protocols may then be used for the expression andpurification of the anti-PD-1 antibodies. For instance, hybridoma clonesmay be grown in serum-free medium and the cell culture broth centrifugedand then filtered. The filtered supernatant containing the antibody maythen be loaded onto an affinity column (e.g., Protein A) column, washedand eluted with an appropriate buffer (e.g., Pierce IgG elute buffer).The eluted fractions may then be pooled and buffer-exchanged into PBS,pH 7.2. The purified antibody may then be analyzed by SDS-PAGE andWestern blot by using standard protocols for molecular weight, yield andpurity. Size exclusion chromatography HPLC may then be performed on anappropriate column (e.g., TSK GEL-G3000 SWXL column (Tosoh)) forbiophysical characterization in order to ensure high antibody purity(generally >90%) with low presence of protein aggregates. Theseprocedures were used in isolating and sequencing nucleic acids encodingSEQ ID NOS. 1-138 from selected hybridoma cells. These techniques,variations thereof, and/or other may also be of use for these purposesas would be understood by those of ordinary skill in the art.

Nucleic acid molecules encoding one or more PD-1 binding agents may becontained within a viral and/or a non-viral vector. In one embodiment, aDNA vector is utilized to deliver nucleic acids encoding one or morePD-1 binding agents to the patient. In doing so, various strategies maybe utilized to improve the efficiency of such mechanisms including, forexample, the use of self-replicating viral replicons (Caley, et al.1999. Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6:723-732; Leitner, et al. 2000. Cancer Res. 60: 51-55), codonoptimization (Liu, et al. 2000. Mol. Ther., 1: 497-500; Dubensky, supra;Huang, et al. 2001. J. Virol. 75: 4947-4951), in vivo electroporation(Widera, et al. 2000. J. Immunol. 164: 4635-3640), incorporation ofnucleic acids encoding co-stimulatory molecules, cytokines and/orchemokines (Xiang, et al. 1995. Immunity, 2: 129-135; Kim, et al. 1998.Eur. J. Immunol., 28: 1089-1103; Iwasaki, et al. 1997. J. Immunol. 158:4591-3601; Sheerlinck, et al. 2001. Vaccine, 19: 2647-2656),incorporation of stimulatory motifs such as CpG (Gurunathan, supra;Leitner, supra), sequences for targeting of the endocytic orubiquitin-processing pathways (Thomson, et al. 1998. J. Virol. 72:2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373),prime-boost regimens (Gurunathan, supra; Sullivan, et al. 2000. Nature,408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al.2001. Science, 292: 69-74), proteasome-sensitive cleavage sites, and theuse of mucosal delivery vectors such as Salmonella (Darji, et al. 1997.Cell, 91: 765-775; Woo, et al. 2001. Vaccine, 19: 2945-2954). Othermethods are known in the art, some of which are described below. Variousviral vectors that have been successfully utilized for introducing anucleic acid to a host include retrovirus, adenovirus, adeno-associatedvirus (AAV), herpes virus, and poxvirus, among others. The vectors maybe constructed using standard recombinant techniques widely available toone skilled in the art. Such techniques may be found in common molecularbiology references such as Molecular Cloning: A Laboratory Manual(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), GeneExpression Technology (Methods in Enzymology, Vol. 185, edited by D.Goeddel, 1991. Academic Press, San Diego, Calif.), and PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.). “Non-viral” plasmid vectors may also be suitable incertain embodiments. Preferred plasmid vectors are compatible withbacterial, insect, and/or mammalian host cells. Such vectors include,for example, PCR-ii, PCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.),pBSii (Stratagene, La Jolla, Calif.), pet15 (Novagen, Madison, Wis.),pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFp-n2 (Clontech, PaloAlto, Calif.), pETI (Bluebacii, Invitrogen), pDSR-alpha (PCT pub. No. WO90/14363) and pFASTBACdual (Gibco-BRL, Grand island, N.Y.) as well asBluescript® plasmid derivatives (a high copy number COLe1-basedphagemid, Stratagene Cloning Systems, La Jolla, Calif.), PCR cloningplasmids designed for cloning TAQ-amplified PCR products (e.g., TOPO™ TAcloning® kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad,Calif.). Bacterial vectors may also be used. These vectors include, forexample, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, BacilleCalmette Guérin (BCG), and Streptococcus (see for example, WO 88/6626;WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376). Many othernon-viral plasmid expression vectors and systems are known in the artand may be use. Other delivery techniques may also suffice including,for example, DNA-ligand complexes, adenovirus-ligand-DNA complexes,direct injection of DNA, CaPO₄ precipitation, gene gun techniques,electroporation, and colloidal dispersion systems. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. The preferred colloidal systemis a liposome, which are artificial membrane vesicles useful as deliveryvehicles in vitro and in vivo. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley, R., et al., 1981, Trends Biochem.Sci., 6: 77). The composition of the liposome is usually a combinationof phospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. Examples of lipids useful in liposomeproduction include phosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

A cultured cell comprising the vector is also provided. The culturedcell may be a cultured cell transfected with the vector or a progeny ofthe cell, wherein the cell expresses the immunogenic polypeptide.Suitable cell lines are known to those of skill in the art and arecommercially available, for example, through the American Type CultureCollection (ATCC). The transfected cells can be used in a method ofproducing an immunogenic polypeptide. The method comprises culturing acell comprising the vector under conditions that allow expression of theimmunogenic polypeptide, optionally under the control of an expressionsequence. The immunogenic polypeptide can be isolated from the cell orthe culture medium using standard protein purification methods.

The skilled artisan has many suitable techniques for using the bindingagents (e.g., antibodies) described herein to identify biologicalsamples containing proteins that bind thereto. For instance, antibodiesmay be utilized to isolate PD-1 using, for example, immunoprecipitationor other capture-type assay. This well-known technique is performed byattaching the antibody to a solid support or chromatographic material(e.g., a bead coated with Protein A, Protein G and/or Protein L). Thebound antibody is then introduced into a solution either containing orbelieved to contain the PD-1 (e.g., an HIV-infected T cell lysate). PD-1may then bind to the antibody and non-binding materials are washed awayunder conditions in which the PD-1 remains bound to the antibody. Thebound protein may then be separated from the antibody and analyzed asdesired. Similar methods for isolating a protein using an antibody arewell-known in the art. The binding agents (e.g., antibodies) may also beutilized to detect PD-1 within a biological sample. For instance, theantibodies may be used in assays such as, for example, flow cytometricanalysis, ELISA, immunoblotting (e.g., western blot), in situ detection,immunocytochemistry, and/or immunhistochemistry. Methods of carrying outsuch assays are well-known in the art.

The binding agents described herein may be also be used to determine thepresence of a disease state in a patient, to predict prognosis, or todetermine the effectiveness of a chemotherapeutic or other treatmentregimen. Expression profile assays, performed as described herein or asis otherwise known in the art, may be used to determine the relativelevel of expression of PD-1. The level of expression may then becorrelated with base (e.g., control) levels to determine whether aparticular disease is present within the patient, the patient'sprognosis, or whether a particular treatment regimen is effective. Forexample, if the patient is being treated with a particularanti-infective regimen, an increased or decreased level of expression ofPD-1 in the patient's tissues (e.g., in peripheral blood, breast tissuebiopsy) may indicate the regimen is worsening or improving the load ofthe infectious agent in that host. The increase or decrease inexpression may indicate the regimen is having or not having the desiredeffect and another therapeutic modality may therefore be selected.

It is also possible to use the binding agents described herein asreagents in drug screening assays to test, for example, new drugcandidates. The reagents may be used to ascertain the effect of a drugcandidate on the expression of the immunogenic target in a cell line, ora cell or tissue of a patient. The expression profiling technique may becombined with high throughput screening techniques to allow rapididentification of useful compounds and monitor the effectiveness oftreatment with a drug candidate (see, for example, Zlokarnik, et al.,Science 279, 84-8 (1998)). Drug candidates may be chemical compounds,nucleic acids, proteins, antibodies, or derivatives therefrom, whethernaturally occurring or synthetically derived. Drug candidates thusidentified may be utilized, among other uses, as pharmaceuticalcompositions for administration to patients or for use in furtherscreening assays.

In some embodiments, the binding agents are in purified form. A“purified” binding agent (e.g., antibody) may be one that is separatedfrom at least about 50% of the proteins and/or other components withwhich it is initially found (e.g., as part of a hybridoma supernatant orascites preparation in the case of a monoclonal antibody). A purifiedbinding agent (e.g., antibody) may be one that is separated from atleast about 50%, 60%, 75%, 90%, or 95% of the proteins and/or othercomponents with which it is initially found.

The polypeptides and nucleic acids described herein may be combined withone or more pharmaceutically acceptable carriers prior to administrationto a host. A pharmaceutically acceptable carrier is a material that isnot biologically or otherwise undesirable, e.g., the material may beadministered to a subject, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art. Suitablepharmaceutical carriers and their formulations are described in, forexample, Remington's: The Science and Practice of Pharmacy, 21^(st)Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005).Typically, an appropriate amount of a pharmaceutically-acceptable saltis used in the formulation to render the formulation isotonic. Examplesof the pharmaceutically-acceptable carriers include, but are not limitedto, sterile water, saline, buffered solutions like Ringer's solution,and dextrose solution. The pH of the solution is generally from about 5to about 8 or from about 7 to about 7.5. Other carriers includesustained-release preparations such as semipermeable matrices of solidhydrophobic polymers containing polypeptides or fragments thereof.Matrices may be in the form of shaped articles, e.g., films, liposomesor microparticles. It will be apparent to those persons skilled in theart that certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of compositionbeing administered. Carriers are those suitable for administration ofpolypeptides and/or fragments thereof to humans or other subjects.Pharmaceutical compositions may also include carriers, thickeners,diluents, buffers, preservatives, surface active agents, adjuvants,immunostimulants, in addition to the immunogenic polypeptide.Pharmaceutical compositions may also include one or more activeingredients such as antimicrobial agents, antiinflammatory agents andanesthetics. The pharmaceutical composition may be administered orally,parentally, by inhalation spray, rectally, intranodally, or topically indosage unit formulations containing conventional pharmaceuticallyacceptable carriers, adjuvants, and vehicles. The term “pharmaceuticallyacceptable carrier” or “physiologically acceptable carrier” as usedherein refers to one or more formulation materials suitable foraccomplishing or enhancing the delivery of a nucleic acid, polypeptide,or peptide as a pharmaceutical composition. A “pharmaceuticalcomposition” is a composition comprising a therapeutically effectiveamount of a nucleic acid or polypeptide. The terms “effective amount”and “therapeutically effective amount” each refer to the amount of abinding agent, nucleic acid or the like used to observe the desiredtherapeutic effect (e.g., restore T cell function).

Methods for treating one or more disease conditions (e.g., HIV orcancer) in a mammalian host comprising administering to the mammal atleast one or more effective doses of one or more binding agents (and/orderivative(s) thereof) described herein are also provided. In someembodiments, the binding agent is a monoclonal antibody or fragment orderivative thereof comprising one or more of SEQ ID NOS. 1-138 and/orshown in Tables 1A and 1B. The one or more binding agents may beadministered in a dosage amount of about 1 to about 50 mg/kg, about 1 toabout 30 mg/kg, or about 5 to about 30 mg/kg (e.g., about any of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, or 40 mg/kg). In certainembodiments, the one or more binding agents may be administered to themammal (e.g., intradermally, intravenously, orally, rectally) at about10 mg/kg one or more times. When multiple doses are administered, thedoses may comprise about the same or different amount of binding agentin each dose. The doses may also be separated in time from one anotherby the same or different intervals. For instance, the doses may beseparated by about any of 6, 12, 24, 36, 48, 60, 72, 84, or 96 hours,one week, two weeks, three weeks, one month, two months, three months,four months, five months, six months, seven months, eight months, ninemonths, 10 months, 11 months, 12 months, 1.5 years, 2 years, 3 years, 4years, 5 years, or any time period before, after, and/or between any ofthese time periods. In some embodiments, the binding agents may beadministered in conjunction with other agents (e.g., anti-infectiveagents and/or chemotherapeutic agent). Such other agents may beadministered about simultaneously with the binding agents, or at adifferent time and/or frequency. Other embodiments of such methods mayalso be appropriate as could be readily determined by one of ordinaryskill in the art.

To assist the skilled artisan in using the antibodies described herein,the same may be provided in kit format. A kit including such antibodiesand optionally other components necessary for using the antibodies todetect cells expressing PD-1 is provided. The antibodies of the kit maybe provided in any suitable form, including frozen, lyophilized, or in apharmaceutically acceptable buffer such as TBS or PBS. The kit may alsoinclude other reagents required for utilization of the antibodies invitro or in vivo such as buffers (e.g., TBS, PBS), blocking agents(solutions including nonfat dry milk, normal sera, Tween-20 Detergent,BSA, or casein), and/or detection reagents (e.g., goat anti-mouse IgGbiotin, streptavidin-HRP conjugates, allophycocyanin, B-phycoerythrin,R-phycoerythrin, peroxidase, detectable labels, and other labels and/orstaining kits (e.g., ABC Staining Kit, Pierce)). The kits may alsoinclude other reagents and/or instructions for using the antibodies incommonly utilized assays described above such as, for example, flowcytometric analysis, ELISA, immunoblotting (e.g., western blot), in situdetection, immunocytochemistry, immunhistochemistry. In one embodiment,the kit provides a binding agent in purified form. In anotherembodiment, the binding agent may be provided in biotinylated formeither alone or along with an avidin-conjugated detection reagent (e.g.,antibody). In another embodiment, the kit includes a binding agentscomprising one or more detectable labels that may be used to directlydetect PD-1. Buffers and the like required for using any of thesesystems are well-known in the art and/or may be prepared by the end-useror provided as a component of the kit. The kit may also include a solidsupport containing positive- and negative-control protein and/or tissuesamples. For example, kits for performing spotting or western blot-typeassays may include control cell or tissue lysates for use in SDS-PAGE ornylon or other membranes containing pre-fixed control samples withadditional space for experimental samples. Kits for visualization ofPD-1 in cells on slides may include pre-formatted slides containingcontrol cell or tissue samples with additional space for experimentalsamples. Other embodiments of kits are also contemplated herein as wouldbe understood by those of ordinary skill in the art.

Thus, this disclosure provides a binding agent that binds PD-1agonistically or antagonistically. In some embodiments, the bindingagent is a polypeptide comprising at least one amino acid sequenceselected from the group consisting of SEQ ID NOS. 1-138 and/or shown inTables 1A and 1B. In some embodiments, the binding agent is apolypeptide comprising one or more combinations of SEQ ID NOS. 1-138(e.g., as shown in Tables 1A and/or 1B). In some embodiments, thebinding agent is an antibody. In some embodiments, the binding agent isa polypeptide such as an antibody comprising a heavy chain CDR1 aminoacid sequence selected from the group consisting of SEQ ID NOS. 1-23. Insome embodiments, the binding agent is a polypeptide such as an antibodycomprising a heavy chain CDR2 amino acid sequence selected from thegroup consisting of SEQ ID NOS. 24-46. In some embodiments, the bindingagent is a polypeptide such as an antibody comprising a heavy chain CDR3amino acid sequence selected from the group consisting of 47-69. In someembodiments, the binding agent is a polypeptide such as an antibodycomprising a light chain CDR1 amino acid sequence selected from thegroup consisting of SEQ ID NOS. 70-92. In some embodiments, the bindingagent is a polypeptide such as an antibody comprising a heavy chain CDR2amino acid sequence selected from the group consisting of SEQ ID NOS.93-115. In some embodiments, the binding agent is a polypeptide such asan antibody comprising a heavy chain CDR3 amino acid sequence selectedfrom the group consisting of 116-138. In some embodiments, the bindingagent comprises the combinations of CDRs shown in Tables 1A and/or 1Band/or has the properties described in Table 2. In some embodiments, thebinding agent is derived from or related to (e.g., by sequence orderivation) a human antibody, human IgG, human IgG1, human IgG2, humanIgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, humanIgA1, human IgA2, human IgD, human IgE, canine antibody, canine IgGA,canine IgGB, canine IgGC, canine IgGD, chicken antibody, chicken IgA,chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, goatantibody, goat IgG, mouse antibody, mouse IgG, pig antibody, and/or ratantibody, and/or a derivative thereof. In some embodiments, thederivative may be selected from the group consisting of an F_(ab),F_(ab2), Fab' single chain antibody, F_(v), single chain, mono-specificantibody, bispecific antibody, trimeric antibody, multi-specificantibody, multivalent antibody, chimeric antibody, canine-human chimericantibody, canine-mouse chimeric antibody, antibody comprising a canineFc, humanized antibody, human antibody, caninized antibody, CDR-graftedantibody, shark antibody, nanobody, and/or canelid antibody. In someembodiments, the binding agent comprises at least a least a first andsecond specificity, the first being against PD-1 and the second beingagainst a different antigen (e.g., an antigen of an infectious agentsuch as HIV (e.g., env) and/or a tumor antigen). In some embodiments,the binding agent and/or derivative thereof may comprise a detectablelabel fixably attached thereto. In some embodiments, the binding agentof any one and/or derivative thereof comprises an effector moiety (e.g.,a cytotoxic drug, toxin, diphtheria A chain, exotoxin A chain, ricin Achain, abrin A chain, curcin, crotin, phenomycin, enomycin, andradiochemical) fixably attached thereto. In some embodiments,polynucleotides encoding one or more binding agents are also provided(e.g., as an expression vector). Host cells comprising and/or expressingthe polypeptide products of such polynucleotides are also provided. Insome embodiments, compositions comprising at least one binding agent orderivative; at least one isolated polynucleotide; at least oneexpression vector; and/or, at least one host cell; or a combinationthereof; and, a pharmaceutically acceptable carrier are also provided.

This disclosure also provides methods for detecting PD-1 on a cell, themethod comprising contacting a test biological sample with a bindingagent or derivative described herein and detecting the binding agentbound to the biological sample or components thereof. Such methods maybe an in vivo method or an in vitro method. In some embodiments, themethod may comprise comparing the amount of binding to the testbiological sample or components thereof to the amount of binding to acontrol biological sample or components thereof, wherein increasedbinding to the test biological sample or components thereof relative tothe control biological sample or components thereof indicates thepresence of a cell expressing PD-1 in the test biological sample (e.g.,mammalian blood). In some embodiments, a system for identifying a PD-1antibody binding agent by assaying the candidate binding agent by theexhaustion functional recovery assay (EFRA); determining the affinity ofthe candidate binding agent for PD-1; and, determining the nucleotidesequence of the CDR of the candidate binding agent is provided.

In some embodiments, a kit for detecting the expression of PD-1 in or ona cell, the kit comprising a binding agent or derivative thereof andinstructions for use. In some embodiments, the binding agent and/orderivative thereof is in lyophilized form.

In some embodiments, this disclosure provides methods for treating,preventing and/or ameliorating an infectious disease, cancer and/orautoimmunity in a mammal comprising administering to the mammal at leastone effective dose of a pharmaceutical composition comprising a bindingagent or derivative thereof. In some embodiments, the infectious diseaseis human immunodeficiency virus (HIV). In some embodiments, the bindingagent and/or derivative thereof used to treat infectious disease and/orcancer is a PD-1 antagonist. In some embodiments, the binding agentand/or derivative thereof used to treat an autoimmune condition is aPD-1 agonist. In some embodiments, multiple doses are administered tothe animal. In some embodiments, the binding agent and/or derivativethereof may be administered in a dosage amount of about 1 to 50 mg/kg.

This disclosure also provides combinations of PD-1 binding agents. Insome embodiments, the combination comprises a first binding agent thatblocks the interaction of PD-1 and PD-L1 and a second binding agent thatdoes not block the interaction of PD-1 and PD-L1. Such combinations maybe used for any use described herein or as may be otherwise ascertainedby those of ordinary skill in the art. For instance, such combinationsmay be used in the methods for treating, preventing and/or amelioratingan infectious disease, cancer and/or autoimmunity in a mammal describedherein.

This disclosure also provides methods for producing the binding agentsdescribed herein by expressing the binding agent in a cell and isolatingthe binding agent from the cell or a culture supernatant of the cell. Insome embodiments, such methods may further comprise expressing a nucleicacid encoding such binding agent(s). In some embodiments, such methodsmay also include combining the binding agent(s) following isolation withone or more pharmaceutically acceptable excipients.

Methods for producing a combination(s) of binding agents, such as afirst binding agent that blocks the interaction of PD-1 and PD-L1 and asecond binding agent that does not block the interaction of PD-1 andPD-L1, are also provided by this disclosure. In some embodiments, thesecond binding agent binds PD-1. In some embodiments, the first and/orsecond binding agents are antibodies such monoclonal antibodies orfragments or derivatives thereof. In some embodiments, the secondbinding agent comprises the amino acid sequences SEQ ID NOS. 2, 25, 48,71, 94 and 117; or SEQ ID NOS. 17, 40, 63, 86, 109, and 132. In someembodiments, these methods may further include the addition of apharmaceutically acceptable excipient.

The terms “about”, “approximately”, and the like, when preceding a listof numerical values or range, refer to each individual value in the listor range independently as if each individual value in the list or rangewas immediately preceded by that term. The terms mean that the values towhich the same refer are exactly, close to, or similar thereto.

As used herein, a subject or a host is meant to be an individual. Thesubject can include domesticated animals, such as cats and dogs,livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratoryanimals (e.g., mice, rabbits, rats, guinea pigs) and birds. In oneaspect, the subject is a mammal such as a primate or a human.

Optional or optionally means that the subsequently described event orcircumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase optionally the composition cancomprise a combination means that the composition may comprise acombination of different molecules or may not include a combination suchthat the description includes both the combination and the absence ofthe combination (i.e., individual members of the combination).

Ranges may be expressed herein as from about one particular value,and/or to about another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent about or approximately, it willbe understood that the particular value forms another aspect. It will befurther understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. Ranges (e.g., 90-100%) are meant to include therange per se as well as each independent value within the range as ifeach value was individually listed.

The term “combined” or “in combination” or “in conjunction” may refer toa physical combination of agents that are administered together or theuse of two or more agents in a regimen (e.g., administered separately,physically and/or in time) for treating, preventing and/or amelioratinga particular disease.

When the terms treat, prevent, and/or ameliorate or derivatives thereofare used herein in connection with a given treatment for a givencondition (e.g., preventing cancer infection by HIV), it is meant toconvey that the treated patient either does not develop a clinicallyobservable level of the condition at all, or develops it more slowlyand/or to a lesser degree than he/she would have absent the treatment.These terms are not limited solely to a situation in which the patientexperiences no aspect of the condition whatsoever. For example, atreatment will be said to have prevented the condition if it is givenduring exposure of a patient to a stimulus that would have been expectedto produce a given manifestation of the condition, and results in thepatient's experiencing fewer and/or milder symptoms of the conditionthan otherwise expected. For instance, a treatment can “prevent”infection by resulting in the patient's displaying only mild overtsymptoms of the infection; it does not imply that there must have beenno penetration of any cell by the infecting microorganism.

Similarly, reduce, reducing, and reduction as used herein in connectionwith prevention, treatment and/or amelioration of a given condition by aparticular treatment typically refers to a subject developing aninfection more slowly or to a lesser degree as compared to a control orbasal level of developing an infection in the absence of a treatment(e.g., administration of one or more PD-1 binding agents). A reductionin the risk of infection may result in the patient's displaying onlymild overt symptoms of the infection or delayed symptoms of infection;it does not imply that there must have been no penetration of any cellby the infecting microorganism.

All references cited within this disclosure are hereby incorporated byreference in their entirety. Certain embodiments are further describedin the following examples. These embodiments are provided as examplesonly and are not intended to limit the scope of the claims in any way.

EXAMPLES Example 1 Generation and Characterization of PD-1 BindingAgents

Four mice strains (total of 16 mice) have been immunized with two PD-1proteins, i.e. human PD-1 Fc fusion protein and a human PD-1 monomericprotein. Mice showing serum reactivity to PD-1 expressed on activatedhuman T lymphocytes have been selected for generation of anti-PD-1hybridoma cell lines. A total of 240 PD-1 hybridoma cell lines wereselected for producing antibodies that bind to recombinant PD-1 protein.The primary criteria for the first round of antibodies selection were:i) staining of PD-1 on activated human T lymphocytes by flow cytometry;ii) diversity of CDR VH and VL sequences as compared to those of theexisting anti-PD-1 antibodies; and, iii) epitope mapping performed bycompetitive binding studies with PD-1 conjugated Luminex beads with twocommercially available anti-PD-1 antibodies binding to differentepitopes on PD-1. A second round of selection was then carried out by:iv) affinity binding assays (not a primary criteria as it does notcorrelate with the stimulatory potential of anti-PD-1 antibodies); v)evaluation of anti-PD-1 antibodies that bind PD-1 and are eithercompetitive, partially competitive or non-competitive with the bindingof PD-L1 in a Luminex biochemical assay; and, vi) functionalcharacterization of antibodies as agonist (not able to restore T-cellsfrom functional exhaustion) or antagonist (able to restore T-cells fromfunctional exhaustion). In these studies, the antibodies were tested anddifferentiated based on their ability to rescue proliferation inHIV-specific exhausted CD8 T-cells.

In a primary screen of antibody supernatants from individual cellcultured hybridoma cell clones, the EFRA assay was carried out toevaluate the functional effect of anti-PD1 antibodies on theproliferation of HIV-specific CD8 T cells (FIG. 2). Antibody clones inthe upper box (E8-3, C2-3, E1-3, F3-3, H8-3, C10-2, G2-1, G3-2, H2-1,and H4-2) act as PD-1 antagonists and stimulate proliferation whileantibody clones in the lower box (C8-1 and G10-2) are agonistic andpromote the PD-1 negative regulatory effect. The level of proliferationinduced by the peptide control (Pep 8) is indicated by the lowerhorizontal line (just below 1%) and the induced proliferation by theMerck MK-3475 anti-PD1 antibody is shown in the upper horizontal line(just above 2%). Antibodies of interest identified by these processesunderwent a second round of subcloning and the resulting hybridomaclones were used for the production and purification of the antibodiesin Table 2. Binding assays were carried out with the purified anti-PD-1antibodies to ensure that the subclones retained their affinity forPD-1. The concentration response binding of anti-PD-1 antibodies to cellsurface PD-1 was evaluated on activated CD4 T-cells (FIG. 3).

The EFRA provides for the selection of binding agents that restoreT-cells from functional exhaustion but are not necessarily antagonistic,meaning those binding agents that do not necessarily interfere with theinteraction between PD-1 and its biological ligand(s) (e.g., PD-L1 orPD-L2). An embodiment of the EFRA was to identify such binding agents(antibodies) that bind PD-1. Epitope mapping of antibody binding to PD-1was performed with two separate biochemical assays. In one assay,competitive binding to PD-1 Fc fusion protein labeled beads wasevaluated between one of two commercial anti-PD-1 antibodies (BMS-5C4and MK3475) and the anti-PD-1 antibodies listed (also described in Table2). Four classes of monoclonal antibodies binding to distinct epitopeswere identified based on this assay. These are: class 1 (competitivewith the EH12.2H7 commercial monoclonal antibody clone that blocks theinteraction of PD-1 with PD-L1), class 2 (competitive with the J116commercial monoclonal antibody clone that binds PD-1 but does noteffectively block the interaction of PD-1 with PD-L1), class 3(competitive with both the EH12.2H7 and J116 commercial monoclonalantibodies), and class 4 (non-competitive with either the EH12.2H7 orJ116 commercial antibodies). The relative binding of these antibodiesfor cell surface PD-1 is represented by the mean fluorescence intensity(MFI) relative to a control anti-PD-1 antibody in FIG. 4. These resultsshow that tight binding antibodies were identified from all four bindingclasses. A second Luminex binding assay was used to directly evaluate ifan anti-PD-1 antibody blocks the interaction between PD-1 and PD-L1.This assay was carried out using PD-1 Fc fusion protein coated beadsthat were incubated in the absence or presence of differentconcentrations of the anti-PD-1 antibodies listed in Table 2. A fixedconcentration of biotinylated PD-L1, approximately equivalent to theIC₅₀ of the PD-1/PD-L1 interaction, was then incubated with thePD-1/antibody complex and PD-L1 binding was detected using fluorescentlylabeled streptavidin (representative binding curves shown in FIG. 5a-c). Using this biochemical assay, antibodies were defined as beingcompetitive, partially competitive or non-competitive with thePD-1/PD-L1 interaction. The competitive, partially competitive andnon-competitive antibodies were identified which bind distinct sites onPD-1 and exhibit high binding affinity (Table 2). Further evaluation ofantibodies that bind PD-1 and are non-competitive or partiallycompetitive with PD-L1 were performed by incubating PD-1 Fc fusionprotein coated Luminex beads with 20 nM of an anti-PD-1 antibody fromTable 2 followed by an incubation of the PD-1/antibody complex withdifferent concentrations of biotinylated PD-L1. FIG. 5d -f shows that acompetitive anti-PD-1 antibody completely blocks the interaction betweenPD-1 and PD-L1. Partially competitive and non-competitive anti-PD-1antibodies listed in Table 2 results in a shift in the binding affinityof PD-L1 for PD-1, but does not block the interaction when concentrationof PD-L1 are increased. Some of these antibodies were also shown toexhibit statistically significant increases in proliferation relative tocontrols (peptide 8 alone control or the peptide and IgG1 isotypecontrol antibody in FIGS. 6a and 6b ). Class 1 antibodies (competitivewith the EH12.2H7 commercial monoclonal antibody that blocks theinteraction of PD-1 with PD-L1) or competitive in the PD-1/PD-L1intereaction assay were generally determined to provide improvedproliferative restoration. In combining the data from multiple EFRAexperiments and using MK-3475 as the common comparator, FIG. 7 showsthat select antibodies described in Table 2 exhibited equivalent orstatistically improved activity (p<0.007) compared to the benchmarkMK-3475 antibody.

Example 2 Antibody Combinations

Combinations of antibodies binding to different PD-1 epitopes were foundto enhance restoration of HIV peptide specific CD8 T-cell proliferationin a functional exhaustion recovery assay (FIG. 8). Synergy betweenantibody types was also observed. For instance, it was determined thatclass 1 (MK-3475 in FIG. 8) and class 2 (139D6 in FIG. 8) antibodies areable to simultaneously bind PD-1. While the maximum stimulation observedfor MK-3475 is consistently about 200% relative to the HIV peptide,combinations of MK-3475 and 139D6 monoclonal antibodies at 5 μg/mlexhibited synergy with a 288% increase in HIV-specific CD8 T cellproliferation relative to the HIV peptide control alone or a 144%increased proliferation relative to MK-3475 or 139D6 added alone (FIG.8). This synergistic increase in proliferation was observed in severalexperimental tests with a statistically significant p value of 0.007. Asa comparison, addition of 10 μg/ml of either MK-3475 or 139D6 alone didnot result in an increased proliferation in the EFRA. Thus, thecombination of a first binding agent that blocks the interaction of PD-1and PD-L1 with a second binding agent that does not block theinteraction of PD-1 and PD-L1 has been determined to act synergisticallyto rescue T cells from exhaustion.

While certain embodiments have been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the following claims.

REFERENCES

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1-54. (canceled)
 55. A combination of binding agents, the combinationcomprising a first binding agent that blocks the interaction of PD-1 andPD-L1 and a second binding agent that does not block the interaction ofPD-1 and PD-L1.